Quantitative reverse transcriptase/PCR assay for the measurement of induction in cultured hepatocytes

Mattes, William B.; Li, Albert P.

doi:10.1016/s0009-2797(97)00073-2

Cited by 17 publications

(13 citation statements)

References 26 publications

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“…Phenobarbital and rifampin previously have been reported to have no effect on CYP1A2 (Mattes and Li, 1997;Meunier et al, 2000;Runge et al, 2000). Our data suggest that both compounds may have the ability to weakly induce CYP1A2 (as measured by EROD activity); although this induction was significant only when ␤-naphthoflavone was removed from the statistical analysis.…”

Section: P450 Induction In Human Hepatocytesmentioning

confidence: 49%

“…These findings are consistent with a study in Cynomolgus monkey, in which CYP2A6 (coumarin 7-hydroxylase) was induced by phenobarbital, but suppressed by ␤-naphthoflavone (Bullock et al, 1995). This observation of induction by rifampin supported by studies in which rifampin was found to induce CYP2A6 (Mattes and Li, 1997;Pichard-Garcia et al, 2000;Rae et al, 2001).…”

Section: Fig 4 Effect Of Treating Primary Cultures Of Human Hepatocmentioning

confidence: 59%

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Effects of Prototypical Microsomal Enzyme Inducers on Cytochrome P450 Expression in Cultured Human Hepatocytes

Madan¹,

Graham²,

Carroll³

et al. 2003

Drug Metab Dispos

305

203

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ABSTRACT:Cultured human hepatocytes are a valuable in vitro system for evaluating new molecular entities as inducers of cytochrome P450 (P450) enzymes. The present study summarizes data obtained from 62 preparations of cultured human hepatocytes that were treated with vehicles (saline or dimethylsulfoxide, 0.1%), ␤-naphthoflavone (33 M), phenobarbital (100 or 250 M), isoniazid (100 M) and/or rifampin (20 or 50 M), and examined for the expression of P450 enzymes based on microsomal activity toward marker substrates, or in the case of CYP2C8, the level of immunoreactive protein. The results show that CYP1A2 activity was markedly induced by ␤-naphthoflavone (on average 13-fold, n ‫؍‬ 28 preparations), and weakly induced by phenobarbital (1.9-fold, n ‫؍‬ 25) and rifampin (2.3-fold, n ‫؍‬ 22); CYP2A6 activity tended to be increased with phenobarbital (n ‫؍‬ 7) and rifampin (n ‫؍‬ 3) treatments, but the effects were not statistically significant; CYP2B6 was induced by phenobarbital (6.5-fold, n ‫؍‬ 13) and rifampin (13-fold, n ‫؍‬ 14); CYP2C8 was induced by phenobarbital (4.0-fold, n ‫؍‬ 4) and rifampin (5.2-fold, n ‫؍‬ 4); CYP2C9 was induced by phenobarbital (1.8-fold, n ‫؍‬ 14) and rifampin (3.5-fold, n ‫؍‬ 10); CYP2C19 was markedly induced by rifampin (37-fold, n ‫؍‬ 10), but relatively modestly by phenobarbital (7-fold, n ‫؍‬ 9); CYP2D6 was not significantly induced by phenobarbital (n ‫؍‬ 5) or rifampin (n ‫؍‬ 5); CYP2E1 was induced by phenobarbital (1.7-fold, n ‫؍‬ 5), rifampin (2.2-fold, n ‫؍‬ 5), and isoniazid (2.3-fold, n ‫؍‬ 5); and, CYP3A4 was induced by phenobarbital (3.3-fold, n ‫؍‬ 42) and rifampin (10-fold, n ‫؍‬ 61), but not by ␤-naphthoflavone. Based on these observations, we generalize that ␤-naphthoflavone induces CYP1A2 and isoniazid induces CYP2E1, whereas rifampin and, to a lesser extent phenobarbital, tend to significantly and consistently induce enzymes of the CYP2A, CYP2B, CYP2C, CYP2E, and CYP3A subfamilies but not the 2D subfamily. Drugs and NMEs5 are often screened for their ability to induce P450 and other drug-metabolizing enzymes with the aim of predicting or explaining drug-drug interactions and pharmacokinetic tolerance.

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Section: P450 Induction In Human Hepatocytesmentioning

confidence: 49%

Section: Fig 4 Effect Of Treating Primary Cultures Of Human Hepatocmentioning

confidence: 59%

Effects of Prototypical Microsomal Enzyme Inducers on Cytochrome P450 Expression in Cultured Human Hepatocytes

Madan¹,

Graham²,

Carroll³

et al. 2003

Drug Metab Dispos

305

203

View full text Add to dashboard Cite

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“…A few drugs have been shown to induce CYP2A6 in human primary hepatocyte culture. These include prototypical inducers rifampicin, dexamethasone, and phenobarbital, although there is wide interindividual variability in response (Maurice et al, 1991;Dalet-Beluche et al, 1992;Mattes and Li, 1997;Meunier et al, 2000;Robertson et al, 2000;Rae et al, 2001;Edwards et al, 2003;Madan et al, 2003). Pyrazole is an inducer of CYP2A6 in vitro (Donato et al, 2000).…”

Section: Nicotine Metabolismmentioning

confidence: 99%

Metabolism and Disposition Kinetics of Nicotine

2005

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“…In order to evaluate the expression of CYP isoforms, the Western blot method and the Northern blot method are widely employed for the determination of CYP content and mRNA content, respectively. Vari ous procedures for the determination of CYP molecu lar mRNA content have been reported to date, 4,5) but these are not suitable for highthroughput measure ment of samples because they are very timeconsum ing processes. Recently, the mouse liver 6) and cul tured human hepatocytes 7) have been reported to pro vide higher sensitivity in the determination of CYP isoform mRNA content using reverse transcription PCR (RTPCR).…”

Section: Introductionmentioning

confidence: 99%

Tissue Distribution of mRNA Expression of Human Cytochrome P450 Isoforms Assessedby High-Sensitivity Real-Time Reverse Transcription PCR

et al. 2003

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Pairs of forward and reverse primers and TaqMan probes specic to each human cytochrome P450 isoform were prepared. Analysis of the mRNA level of each CYP isoform in total RNA from pooled specimens of various human or gans was performed by realtime reverse transcription PCR using an ABI PRISM 7700 sequence detector system. The ex pression of CYP3A4 mRNA was similar to that of CYP3A7 mRNA in the fetal liver, and CYP3A4 mRNA levels in the fetal liver were about 0.1 times lower than in the adult liver. CYP2E1 showed the highest level of mRNA expression in the liver. The mRNA expression of 30 CYP isoforms (CYP1A1, 1A2, 1B1, 2A6, 2A7, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 2J2, 3A4, 3A5, 3A7, 4A11, 4F2, 4F3, 5A1, 7B1, 8A1, 8B1, 17, 26A1, 27, 27B1, 39A1, 46, and 51) in the liver was successfully detected by this method. CYP2F1, 4B1, 4F8, 11s (11A, 11B1, and 11B2), 19, and 24 mRNA levels were the highest in the lung, lung, prostate, adrenal gland, placenta, and kidney, respectively; however, the mRNA expression of these eight CYP isoforms in the liver was not detected by this method. The mRNA levels of the CYP isoforms deter mined in various human tissues were in good agreement with previously reported data. The method described here has the advantages of high specicity and excellent quantication over a wide range of mRNA concentrations, making it suitable for the evaluation of a large number of samples in the assessment of the expression prole of drugmetabolizing enzymes.

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Quantitative reverse transcriptase/PCR assay for the measurement of induction in cultured hepatocytes

Cited by 17 publications

References 26 publications

Effects of Prototypical Microsomal Enzyme Inducers on Cytochrome P450 Expression in Cultured Human Hepatocytes

Effects of Prototypical Microsomal Enzyme Inducers on Cytochrome P450 Expression in Cultured Human Hepatocytes

Metabolism and Disposition Kinetics of Nicotine

Tissue Distribution of mRNA Expression of Human Cytochrome P450 Isoforms Assessedby High-Sensitivity Real-Time Reverse Transcription PCR

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